Project Summary The rising incidence of multi-drug- and extensively drug-resistance strains of Mycobacterium tuberculosis (Mtb), which causes tuberculosis (TB), threatens the efficacy of our current therapeutic arsenal. Thus, the WHO strategic plan to eliminate TB emphasizes the development of new drugs. Processes within the cell envelope are attractive as potential novel drug targets because Mtb modulates metabolic activities within the cell envelope in response to environmental changes during infection, and these adaptations are essential to Mtb viability and pathogenesis. However, our exploration of cell envelope pathways is constrained by our limited knowledge of protein localization and function in the cell envelope and the lack of tools with which to interrogate them. The objective of this application is to overcome this obstacle by developing a labeling methodology that can be targeted to specific subcellular compartments, including the cell envelope. The rationale is that new tools to study protein localization and interactions will allow us to, for the first time, (1) accurately inventory the cell envelope proteome and (2) interrogate protein-protein interactions in the cell envelope. Our innovation is to apply a live-cell protein labeling method that has not, to our knowledge, been applied to mycobacteria. In contrast to existing methods, this approach preserves cellular architecture and native protein interactions. From our preliminary studies we have evidence that the method will be successful in our achieving our goals in mycobacteria. We therefore propose to characterize the non-canonical secreted proteome of Mtb and elucidate the interactome of a putative non-canonical secreted protein that is essential for cell growth in division. As a result of these studies we will have novel techniques to study cell envelope proteins and will gain insight into protein localization and interactions that contribute to Mtb growth and adaptation. These results are anticipated to have a positive impact on the evaluation of cell envelope proteins as targets for anti-tuberculosis therapy and to advance our fundamental knowledge of mycobacterial cell envelope processes and pathways.